Nuclear factor of activated T-cells 5 (NFAT5, TonEBP, or OREBP), is a human gene that encodes a transcription factor that regulates the expression of genes involved in the osmotic stress.
The NFAT5 protein is basically expressed in all tissues and cellular types, particularly in tissues that are often subjected to osmotic stresses, such as kidneys, eyes, and skin. The molecular mechanism of NFAT5 response in osmotic stress is well known, but NFAT5 is also involved in other biological roles, such as in embryonic development, in integrin-induced cell migration, in cellular proliferation. The mechanism by which NFAT5 acts in these other processes is currently not well known.
The NFAT5 (Nuclear factor of activated T-cells 5) has been identified as the transcription factor necessary for survival of renal cells in the challenging conditions of renal medulla (Miyakawa H, 1999; Woo S K, 2000b). The NFAT5 protein is highly expressed in renal medulla (Sykes, 2007). Knockout by disruption of both alleles in mice is typically embryonically lethal, and surviving NFAT5-null mice have profound and progressive atrophy of the renal medulla (Lopez-Rodriguez C, 2004). Transgenic (Tg) mice that overexpress OREBPdn (dominant negative form of TonEBP) specifically in the epithelial cells of the renal collecting tubules have impaired ability to concentrate urine, and show progressive atrophy of the renal medulla and cortical thinning, which in the most severe cases is accompanied by severe hydronephrosis and loss of medullary structure (Lam A, 2004).
NFAT5 is a member of the Rel family of transcriptional activators, which includes nuclear factor κB (NFκB) and the nuclear factor of activated T-cells (NFAT). Hypertonicity increases NFAT5 mRNA in MDCK (Woo S, 2000b), HeLa (Ko B C, 2000) and mouse inner medullary collecting duct (mIMCD3) cells (Cai Q, 2005). Hypertonicity transiently increases NFAT5 mRNA and protein abundance (Woo S, 2000a), peaking 4-12 h after hypertonicity depending on cell type. At 300 mOsm, NFAT5 is present in the nucleus and cytoplasm, but after hypertonicity it moves rapidly into the nucleus, inducing its transcriptional activity. In the rat kidney, nuclear localization of NFAT5 is decreased after water loading and increased after dehydration (Cha J H, 2001). The NFAT5 target gene contains at least one osmotic response element (ORE) consensus (Ferraris J, 1994; Ferraris J, 1996; Ferraris J, 1999; Woo S, 2000a; Dahl S C, 2001) and AP-1 site (Irarrazabal C, 2008). Additionally, high NaCl increases NFAT5 transactivating activity (Ferraris J, 2002a; Irarrazabal C E, 2010). NFAT5 activation by hypertonic stress results in the induction of several genes implicated in osmotic tolerance, such as aldose reductase (AR) (Burg M, 2007).
There are positive and negative (Colla E, 2006; Chen Y, 2007) upstream molecular regulators of the tonicity-dependent activation of NFAT5 transactivating activity. Positive regulators include: cAMP-dependent kinase (PKA) (Ferraris J, 2002b); p38 mitogen-activated protein kinase (MAPK) (Ko B C, 2002); Fyn, a member of the SRC family of non-receptor, cytoplasmic protein tyrosine kinases (Ko B C, 2002); Ataxia Telangiectasia Mutated (ATM) (Irarrazabal C, 2004, Zhang Z, 2005); phosphatidyl 3-kinase Class IA (PI3K-IA) (Irarrazabal C, 2006) and PLCγ1 (Irarrazabal 2010). Experiments using HEK293 and Jurkat cells demonstrate that the PI3-K class IA is upstream of ATM in high NaCl-induced activation of NFAT5 (Irarrazabal C, 2006).
The current paradigm postulates that NFAT5 presents tonicity-dependent activation dependent on oxidative stress: antioxidants that reduce ROS suppress high NaCl-induced activation of NFAT5 transcriptional activity (Zhou X, 2005; Zhou X, 2006). The urine concentrating mechanism in the kidney implies an increased osmolality, associated with low Pa02, allowing reactive oxygen species (ROS) to increase (Rosas-Rodríguez J A, 2010). On the other hand, there is evidence suggesting that ATM (NFAT5 regulator) is activated during hypoxia and hypoxia-reoxygenation in cancer cells (Hammond E M, 2004; Bencokova Z, 2009). However there is no information about the effect of hypoxia on NFAT5. We hypothesize that low oxygen concentration could induce NFAT5 activation. To study the effect of low oxygen on NFAT5 expression and activity we used primary cultures of rat IMCD and HEK293 cells, grown in isotonic and hypertonic media. We also analyzed the effect of hypoxia on cell death when NFAT5 was knocked down. Additionally, we tested the in vivo effect of hypoxia on NFAT5 activity in an experimental model of renal ischemia/reperfusion (I/R) in the rat. In the I/R kidneys we measured the mRNA and protein abundance of NFAT5, one of its downstream genes (aldose reductase, AR) and two of its upstream activators (ATM and PI3K). Our results show that NFAT5 is activated in vitro and in vivo by hypoxia and ischemia/reperfusion.
Myocardial infarction (MI) is the major cause of death and disability worldwide and continues to be a major public health problem despite considerable advances in diagnosis and management over the last decades (Thygesen K, 2007). The main determinants of patient outcome following MI are myocardial infarct size and left ventricular (LV) remodelling. Whereas infarct size is determined in the acute phase following MI, LV remodelling is a chronic maladaptive process, characterized by myocardial hypertrophy, fibrosis, progressive ventricular dilatation, and deterioration of cardiac performance over time, which eventually leads to congestive heart failure. One of the elements involved in the ischemic condition of MI is the hypoxia and there is not information about the role of NFAT5 during MI.
In general, the adult central nervous system (CNS) possesses a limited capacity for regeneration after injury, including ischemia. Following ischemic injury, neural tissue recovery is accompanied by the formation of reactive astrogliosis; this process is vital for isolating necrotic tissue from its uninjured surroundings, but concurrently, it markedly impedes regenerative processes. Shortly after ischemia, a series of ionic, neurotransmitter and oxidative radical imbalances occurs that lead to the activation of microglia and subsequently to an increased number of reactive astrocytes. Both cell types release cytokines and other soluble products (Tian D S, 2007) that play an important role in consecutive processes, including the apoptosis of oligodendrocytes (Yang Y, 2011) and neurons (Pettigrew L C, 2008). There is no information about the potential role of NFAT5 during hypoxia in CNS.